Simone Granchi

Identification, localization and functional activity of oxytocin receptors in epididymis

Oxytocin (OT) is a neurohypophysial hormone with unclear physiological functions in the male. Several previous studies indicated that OT might have a role in the ejaculatory process, stimulating sperm release from the epididymal storage. In this study we investigated on the presence and function of OT receptor (OTR) in rabbit and human epididymis. By using RT-PCR, Western and binding studies, we found that OTR gene and protein is expressed in the human epididymis and stimulates in vitro contractility. The immunolocalization of OTR suggests that the receptor is not only present in the smooth muscle cells of the human epididymis but also in the epithelial compartment. Experiments performed in rabbit epididymal epithelial (rEE) cells in culture indicate that OT induces the release of an other potent stimulator of epididymal contractility, endothelin-1 (ET-1), Blocking the ET(A) subtype of the ET-1 receptors, by using a specific antagonist (BQ-123), partially counteracts the contractile effect of OT, suggesting positive interactions between the two peptides in regulating epididymal contractility. Finally, to investigate whether an acute OT administration increases sperm release also in humans, we treated oligozoospermic patients with an intravenous bolus of OT (2.5 IU), just before sperm collection. In a small, single blind study, we found that OT almost doubled sperm retrieval when compared with vehicle administration. Our results indicate that OT might have physiological functions also in the male, controlling epididymal motility and sperm progression through the male genital tract.

Endothelium-dependency of yohimbine-induced corpus cavernosum relaxation.

Development and maintenance of penile erection requires the relaxation of the smooth muscle cells in the cavernous bodies and is essentially mediated by nitric oxide (NO). The penile flaccid state is conversely maintained by the alpha adrenergic neuroeffector system and by other vasoconstrictors, such as endothelin-1 (ET-1). In this study we examined the mechanisms involved in yohimbine-induced relaxation in human and rabbit corpora cavernosa (CC). We essentially found that yohimbine not only blocks contractions induced by adrenergic agonists, but also by non-adrenergic substances, such as ET-1. This effect was unrelated to antagonism at the level of ET receptors, because yohimbine did not affect ET-1-induced increase in intracellular calcium in isolated CC cells. Conversely, our data suggest that yohimbine counteracts ET-1-induced contractions by interfering with NO release from the endothelium. In fact, yohimbine-induced CC relaxation was inhibited by the mechanical removing of the endothelium and by blocking NO formation or signalling via guanylate cyclase and cGMP formation. Conversely, yohimbine activity was strongly increased by inhibiting cGMP degradation. In an experimental model of hypogonadism, performed on rabbits by chronic treatment with a long-lasting GnRH agonist, the relaxant yohimbine activity was also decreased, but completely restored by androgen supplementation. This effect was evident only in preparations in which the main source of NO was present (endothelium) or in which NO formation was not impaired by L-NAME. Our data indicate that the relaxant effect of yohimbine is both endothelium and androgen-dependent. This might justify the lack of efficacy of this drug in treatment of some form of organic erectile dysfunction.

Endothelin-1 is Synthesized and Biologically Active in Human Epididymis via a Paracrine Mode of Action

In a previous study, we reported the presence of endothelin-1 and endothelin receptors in the human testis. We have now extended our investigations to the human epididymis. Since sperm appear to be immotile during their transit through the epididymis, it is conceivable that specific local factors promote smooth muscle contraction, enabling sperm transport. In this paper, we show that endothelin-1 mRNA and protein are readily detectable in the epithelial compartment of the human epididymis, and that endothelin converting enzyme- 1, which converts the precursor pro-endothelin-1 into active endothelin-1, is expressed in the epididymis, consistent with active processing of the prohormone. In addition, two classes of endothelin receptors were characterized and located in the muscle cells of the epididymis. These receptors correspond, in terms of affinity constants and capacity, to the previously characterized endothelinA and endothelinB receptor. These receptors appear to be biologically active and mediate the contractile activity of the epididymis in vitro. Our data suggest that endothelin-1, via a paracrine mode of action, may be responsible for sperm progression through this organ.

Uteroglobin reverts the transformed phenotype in the endometrial adenocarcinoma cell line HEC‐1A by disrupting the metabolic pathways generating platelet‐activating factor

Uteroglobin, originally named blastokinin, is a protein synthesized and secreted by most epithelia, including the endometrium. Uteroglobin has strong anti‐inflammatory properties that appear to be due, at least in part, to its inhibitory effect on the activity of the enzyme phospholipase A2. In addition, recent experimental evidence indicates that uteroglobin exerts antiproliferative and antimetastatic effects in different cancer cells via a membrane receptor. The human endometrial adenocarcinoma cell line HEC‐1A does not express uteroglobin. Thus, we transfected HEC‐1A cells with human uteroglobin cDNA. The transfectants showed a markedly reduced proliferative potential as assessed by impaired plating efficiency as well as by reduced growth in soft agar. Cytofluorimetric analysis clearly indicated that in uteroglobin‐transfected cells the time for completion of the cell cycle was increased. We previously demonstrated that HEC‐1A cells actively synthesize platelet‐activating factor, one of the products of phospholipase A2 activity. In addition, we demonstrated that platelet‐activating factor stimulates the proliferation of these cells through an autocrine loop. In uteroglobin transfectants, the activity of phospholipase A2 and platelet‐activating factor acetyl‐transferase, which are involved in the synthesis of platelet‐activating factor, was significantly reduced compared with wild‐type and vector‐transfected cells (p < 0.05). Our results indicate that enforced expression of uteroglobin in HEC‐1A cells markedly reduced their growth potential and significantly impaired the synthesis of platelet‐activating factor, an autocrine growth factor for these cells. These data suggest that one possible mechanism for the recently observed antineoplastic properties of uteroglobin may be the inhibition of the synthesis of platelet‐activating factor. Int. J. Cancer 88:525–534, 2000.

The effects of an autocrine loop mediated by platelet-activating factor (PAF) in HEC-1A cells are reverted by uteroglobin

Platelet-Activating Factor (PAF), a proinflammatory phospholipid, is produced in high amount by endometrial cells both in basal conditions and following different stimulations”.”. In the same tissue, specilic PAF receptors have been demonstrated”. Several studies have suggested a role for PAF in implantation”, 5, although more recent evidences indicate a prominent role of this phospholipid in angiogenesis‘. We have studied PAF production, expression of PAF receptors and the role of PAF/PAF receptor system in growth processes in the human endometrial adenocarcinoma cell line HEC-lA, which was established by Kuramoto et al, in 1972’. Early studies performed by our group, demonstrated that HEC-lA cells are able to synthesize PAI?” at least through the remodelling pathway of PAF synthesis. Indeed, the activity of PAF acetyltransferase, the main enzyme involved in PAF synthesis through the remodelling pathway together J